Lymphocytes and their precursors contain enzymes that are expressed at varying levels as the cells differentiate. Terminal deoxynucleotidyl transferase (TdT) is a single-strand specific DNA polymerase that is catalytically active at the differentiation state when rearrangements in immunoglobulin and T-cell receptor genes are occurring. The enzyme is thought to be involved in the process. Adenosine deaminase (ADA) is a purine salvage pathway enzyme that is present in very high levels in prelymphocytes. This enzyme activity is essential for a functional immune system; it appears to protect cells from toxic levels of deoxyadenosine. Perturbations in the activity of either TdT or ADA have been shown to have profound effects on lymphoid development. Inhibitors of ADA have been used clinically to modulate immune function. No specific inhibitions of TdT have been identified. At present the catalytic mechanisms of these enzymes are not fully understood and only limited information regarding the structure of substrate binding and catalytic domains is available. We have developed reagents and structural information that allow us to probe the enzyme active sites. The principal strategy we have used for defining the functional domains of these enzymes involves direct site identification through photolabeling and selection of a variety of group specific reagents based on the identities of photolabeled peptides. Photoactive reagents (synthesized by our group) include nucleotide analogs (arylazides), nucleoside analogs and potent ADA inhibitor analogs (arylazides) and novel arylazide containing DNAs. These light sensitive DNAs are potentially excellent tools with which to study DNA binding proteins. Following site identification, a variety of chemical probes will be used to tag specific amino acids within the active site domains. The catalytic mechanism of ADA will be additionally probed by fluorescence studies of ADA bound to a series of potent inhibitors. The interactions between the enzyme and inhibitors will be determined using time-resolved fluorescence analysis (monitoring trp fluorescence) of genetically engineered ADAs containing a single trp at each of the four naturally occurring positions. These studies will lead to precise definition of the active sites of these key enzymes and will suggest strategies for the design of modulators of enzyme activities that may have clinical significance.